Almost all bacteria including human pathogens contain suicide or toxin genes encoded by the toxin-antitoxin (TA) systems, which are induced under various growth and stress conditions leading to cell growth arrest and eventual cell death in a way similar to apoptosis or programmed cell death in higher systems. Surprisingly, many bacteria, particularly human pathogens such as Mycobacterium tuberculosis contain a large number of independent toxin genes on their genomes. The importance of these toxin genes in medical science has not been fully appreciated until most recently when our laboratory and others started to decipher the cellular targets of these toxins and demonstrated how these toxins inhibit cell growth leading to cell death. Bacterial physiology is tightly regulated by these toxins or by the toxin networks under stress conditions. The prolonged dormancy of M. tuberculosis in human tissues, biofilm formation, and persistent multi-drug resistance of various other human pathogens are presumed to be closely associated with toxin expression in these pathogens. Furthermore, taking advantage of suicidal properties of these toxins, it is possible to develop novel unprecedented antibiotics against human pathogens. Therefore, all-out comprehensive investigation in this emerging field is urgently needed. For this reason, I propose to carry out thorough investigation of the toxin-antitoxin systems in Escherichia coli as a model bacterium, which surprisingly contains as many as twenty two independent toxin-antitoxin systems on its genome. As these toxins are functionally highly diversified and yet are conserved in many other bacteria, they are ideal paradigms for the study of not only bacterial toxins, but also previously unknown bacterial physiology governed by the TA network. Our goal is to decipher cellular targets of individual E. coli toxins and their mechanisms of action. I will explore to elucidate how the TA systems are coordinated to from the TA network in the cell. In Aim #1, a combination of genetic, biochemical and structural approaches will be used to decipher new TA systems including identification of cellular targets and mechanism of action of toxins. In Aim #2, with help of TA deletion strains we will investigate possible roles of individual TA systems and a group of the TA systems in bacterial physiology under different growth or stress conditions and how the expression of individual TA systems is coordinated in the overall TA network system. Identification of cell death determinants which will be also carried out in this proposal will provide important insights into the mechanism of bacterial cell death. Relevance in public heath: The present studies will provide new insights into the role of suicide genes present in human bacterial pathogens in their pathogenicity and drug-resistant persistence, providing important clues for development of novel antibiotics targeting the TA systems. PUBLIC HEALTH RELEVANCE: Bacteria, including most of human pathogens, contain the TA (toxin-antitoxin) systems, which regulate their own cell growth and determine their own life and death. Research on the TA Systems has revealed their important roles in persisting drug-resistance and pathogenicity of human pathogens. The proposed search is to characterize all the TA systems in E. coli elucidating cellular targets of individual toxins, their mechanisms of action and their roles in bacterial physiology. The outcome from this research has a wide, important implication to our understanding of the basic principle of bacterial physiology, the roles of the TA systems in human pathogens and provides crucial insights into a novel technology to suppress cell growth of human pathogens or to lead them to death.